Dixit Anjali, Paegel Brian M
Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, CA, USA.
Department of Chemistry, University of California, Irvine, Irvine, CA, USA.
Nat Protoc. 2025 May 22. doi: 10.1038/s41596-025-01190-4.
Solid-phase DNA-encoded library (DEL) synthesis is a next-generation drug discovery technology with powerful activity-based and cellular lead identification capabilities. Solid-phase DELs combine the one-bead-one-compound approach with DNA encoding to furnish beads that display multiple copies of photocleavable library members and DNA encoding tags. Sequential chemical synthesis and enzymatic DNA ligation reactions yield an encoded library in which individual library members are physically isolable, enabling various high-throughput screening modalities. This advancement from on-DNA synthesis, in which small molecules are directly attached to their DNA-encoding tags, decouples the library member from the steric bulk of the DNA tag, which prevents biased binding to a target. Here we provide step-by-step instructions for solid-phase DEL synthesis, incorporating all of our most recent quality control innovations to ensure robust library production. The protocol begins with on-bead synthesis of a linker containing a spectroscopic handle for chromatographic analysis, an ionization enhancer for mass spectrometry and an alkyne for installation of DNA encoding sites via copper-catalyzed azide-alkyne cycloaddition click chemistry. Coupling of a photocleavable linker before library synthesis enables compound liberation from the bead for activity-based screening. Powerful combinatorial split-and-pool parallel synthesis tactics transform modest collections of small-molecule building blocks into large DELs of all possible building block combinations. Post synthesis, decoding and mass analysis of single DEL beads as well as whole-library deep sequencing provides rigorous chemical and bioinformatic quality control and establishes suitability for screening. The solid-phase chemistry is highly accessible: expertise in chemical synthesis is not necessary and solid-phase synthesis apparatus is routinely available in molecular biology laboratories. This procedure requires ~1 month to complete.
固相DNA编码文库（DEL）合成是一种具有强大的基于活性和细胞先导物识别能力的下一代药物发现技术。固相DEL将单珠单化合物方法与DNA编码相结合，以提供展示可光裂解文库成员和DNA编码标签多个拷贝的珠子。连续的化学合成和酶促DNA连接反应产生一个编码文库，其中各个文库成员在物理上是可分离的，从而能够进行各种高通量筛选模式。这种从DNA上合成（小分子直接连接到其DNA编码标签上）的进步，使文库成员与DNA标签的空间体积解耦，这防止了与靶标的偏向性结合。在这里，我们提供了固相DEL合成的分步说明，纳入了我们所有最新的质量控制创新，以确保稳健的文库生产。该方案首先在珠子上合成一个连接子，该连接子包含用于色谱分析的光谱手柄、用于质谱分析的离子化增强剂以及用于通过铜催化的叠氮化物-炔烃环加成点击化学安装DNA编码位点的炔烃。在文库合成之前偶联可光裂解的连接子，能够从珠子上释放化合物以进行基于活性的筛选。强大的组合分割合并平行合成策略将少量小分子构建模块转化为所有可能构建模块组合的大型DEL。合成后，对单个DEL珠子进行解码和质量分析以及对整个文库进行深度测序，提供了严格的化学和生物信息学质量控制，并确定了筛选的适用性。固相化学非常容易操作：不需要化学合成方面的专业知识，并且分子生物学实验室通常可以使用固相合成设备。这个过程大约需要1个月完成。
